HIV-1 displays complex phenotypes for coreceptor usage and repertoire of host cells. Types of cells targeted by HIV-1 can have a profound impact for disease progression, provide different intracellular milieus that can facilitate evasion of immunity, modulation of variants within the viral quasispecies, and provide reservoirs for latent infection. Macrophage tropism by variants of HIV-1 is regulated at the level of entry by viral use and host cell expression of coreceptors CCR5 or CXCR4. Genetic determinants of coreceptor usage map to HIV- 1 env, in particular hypervariable domain V3 in Env gp120. CXCR4-mediated entry can be distinct for different cell types. The rationale for the proposed studies is that CXCR4-using viruses develop in vivo, and contribute to disease progression. The hypothesis is that complex, discontinuous determinants that map to regions of Env outside V3 modulate interactions between Env gp120 and CXCR4 in a cell-type dependent manner. Three Specific Aims are proposed: 1. Map genotypic evolution in vivo of envelope gp120 V1-V5 regions; 2. Map determinants in gp120 that confer D-X4 phenotype in different primary cells; and 3. Determine functional impact of gp120-mediated coreceptor usage on macrophages and thymocytes. Studies are cross-sectional, including primary subtype B, A, and D viruses from peripheral blood, and longitudinal, including viruses from peripheral blood prior to and following antiretroviral therapy and from peripheral blood and tissue compartments. The proposed studies use innovative technologies and longitudinal evaluation of genotype, phenotype, and function of envelopes from individuals with well-defined clinical variables to identify determinants among viruses that emerge in vivo prior to development of late stage disease. The proposed studies are significant for defining unique phenotypic characteristics of envelopes that use CXCR4 for infection of macrophages and thymocytes, immune biomarkers that aid clinicians in defining the optimal timing for initiation of ART in children, development of novel, cell-type specific inhibitors of chemokine receptor interactions, and designing therapeutic vaccines with effects across subtypes and coreceptor usage to prevent evolution of viruses with expanded tropism and pathogenesis.